ISSN 1409 - 8695 UDC: 615.849.2 Original scientiic paper
Introduction
The development of Nuclear Medicine towards molec-ular diagnostic imaging modality in the past two decades was followed by intensive research and applicative work resulting with introduction of wide variety of new genera-tion radiopharmaceuticals. Labeled peptides have proven their potential as targeting agents for receptor scintigraphy, especially the synthetic analogs with preferable biological half-life and in vivo stability. Octreotide, a somatostatin analog, was used to compound 111In-DTPA-octreotide
(Oc-treoScan), the irst commercial radiolabeled peptide, reg -ulatory approved in Europe and USA for clinical applica-tion. It became the imaging agent of choice for detection of
“In-house” preparation of
99mTc-EDDA/HYNIC-TOC, a speciic
targeting agent for somatostatin receptor scintigraphy
Sonja Kuzmanovska*, Olivija Vaskova, Marina Zdraveska Kocovska
Institute of Pathophysiology and Nuclear Medicine, Medical Faculty, University “Sts Cyril and Methodius”, Vodnjanska 17, Skopje, Republic of Macedonia,
Received: November 2011; Accepted: January 2012
Abstract
The use of radiolabeled peptide ligands as diagnostics and therapeutics in nuclear oncology has increased recently. One of the most frequently used radiopharmaceutical is 99mTc-EDDA/HYNIC-TOC, a somatostatin analog with afinity for certain types of somatostatin re -ceptors, overexpressed in tumors of neuroendocrine origin. The radiopharmaceutical is not readily available; therefore we introduced its “in house” preparation within project activities supported by the International Atomic Energy Agency (IAEA). We optimized the radiolabeling protocol, prepared a small batch of frozen kits, performed ITLC quality control and animal biodistribution during the preclinical evaluation procedures. The co-ligand exchange labeling procedure was carried out at 100°C during 10 min, resulting in radiochemical purity >90%. The biodistribution scintigrams in normal Wistar rats showed rapid blood clearance after 15 min and predominant kidney accumulation af-ter 4 h, in accordance with the data reported by other authors. Storage stability of the formulated small batch frozen kit (-20°C) was evalu-ated within 6 months, with radiolabeling yield ranging between 94,3% and 96,9%. We conclude that frozen kit can be a safe alternative to the freeze-dried for small batch in house production, and after the satisfactory preclinical evaluation, the “in house” prepared 99mTc-EDDA/ HYNIC-TOC can be introduced in clinical practice asspeciic targeting agent for somatostatin receptor scintigraphy.
Key words: radiopharmaceutical, technetium-99m, tyr3-octreotide, receptor scintigraphy, in house production, quality control
* skuzmanovska@gmail.com, +389 2 3112831, fax: +389 2 3147203
somatostatin (SST) receptors, overexpressed in neuroen-docrine tumors (NET), with evidence of higher sensitiv-ity in comparison with CT and MR scanning (Shi et al.,
1998). Besides the speciic somatostatin receptor imaging
properties, somatostatin receptor scintigraphy can be used as tumor staging agent (Lebtahi et al., 1997), as well as a predictor of the effectiveness of therapy with somatosta-tin analogues (Krenning et al., 1993). Despite of the fa-vorable features, 111In-DTPA-octreotide has limited
avail-ability, suboptimal imaging gamma energy and high cost. Therefore, efforts were made by different research groups worldwide to replace the radiometal 111In with 99mTc, the
most preferable imaging radionuclide for single photon emission computed scintigraphy (SPECT) up to date (Mai-na et al., 1995; Vallabhajosula et al.,1996; Decristoforo et al.,2000).
molecules and limited number of donor atoms, the indi-rect labeling approach is shown to be the most preferable technique (Liu et al., 1997). Thus, a bifunctional chelator is used to conjugate the peptide molecule and simultane-ously form a coordination bound with the reduced 99m
Tc-pertechnetate. One of the earliest eficient labeling meth -ods was reported by Maecke (Maecke and Béhé, 1996) and later developed by Decristoforo (Decristoforo and Mather, 1999). They used Tyr3octreotide (TOC) conjugated with
HYNIC (hydrazinonicotic acid) as a bifunctional chelator. When using HYNIC as a bifunctional chelator, the com-plex has to be stabilized through the addition of a co-ligand or a mixture of co- ligands. To stabilize the 99mTc -
HYNIC-TOC complex, EDDA (ethylendiamine N, N’ diacetic acid) was used as co-ligand, resulting with favorable biodistribu-tion, low blood levels and high renal excretion of the radio-pharmaceutical.
Since 99mTc-EDDA/HYNIC-TOC was evidenced to
be an eficient tool for detecting SST2 and SST5 recep -tor subtypes, the main targets for the somatostatin recep-tor scintigraphy are carcinoids, insulinomas, pituitary adeno-ma, pheochromocytoadeno-ma, neuroblastoadeno-ma, medulary thyroid carcinoma. The radiopharmaceutical is not readily avail-able, therefore our goal was to introduce it as “in house” product within the project activities supported by the Inter-national Atomic Energy Agency (IAEA), for updating and expanding the range of nuclear oncology services provid-ed by our institute.
Material and methods
- Chemicals with high purity grade were purchased by Merck, Germany (SnCl2 ·2H2O), or granted by IAEA (EDDA from Fluka Chemie Gmbh, Tricine from Sigma-Aldrich Chemie Gmbh).
- The peptide conjugate (HYNIC-TOC) was provided by Radioisotope Centre POLATOM, Poland
- 99mTc-pertechetate was obtained as fresh eluate from
commercial 99Mo/99mTc generator (ELUMATIC-III)
sup-plied by Cis biointernational, France
- For the product puriication SepPak C-18 mini car
-tridges (Waters, Milford, USA) were used, and for the inal sterilization of the radiopharmaceutical 0,20 μ sterile Mil
-lipore ilters
- For the quality control TLC-SG (Merck, 5553 Silica gel 60) aluminum sheets were used
- Biodistribution studies were performed on normal fe-male Wistar rats in accordance with EC Directive 86/609/ EEC for animal experiments
Preparation of the radiopharmaceutical
In order to optimize the radiopharmaceutical produc-tion protocol concerning own facilities two approaches were implemented:
1. Production of a single dose wet labeled 99m
Tc-ED-DA/HYNIC-TOC
2. Production of a small-batch frozen kits for labeling with 99mTc-pertechnetate
The optimized labeling conditions (pH, temperature,
reaction time, speciic activity) applied during the wet la -beling procedure were identical during the la-beling of fro-zen kits.
Wet labeling protocol was conducted according the publication of Decristoforo (Decristoforo and Mather, 1999), via co-ligand exchange as follows: in a sterile glass
vial 20μg of HYNIC-TOC were mixed with 1 mL of so -lution mixture (1:1) consisting of 20 mg EDDA and 10
mg tricine, 15 μg of SnCl2·2H2O dissolved in 0,1 M NCl
and incubated with addition of 1000 MBq
99mTc-pertech-netate (up to inal volume of 2 mL) in water bath at 70
°C for 30 minutes (protocol 1). The solutions were fresh-ly prepared and purged with sterile nitrogen. To optimize the reaction time we followed the von Guggenberg (von
Guggenberg et al., 2003) modiication of the previous la -beling protocol and heated the reaction mixture at 100 °C for 10 minutes (protocol 2). The pH of the labeled prod-uct was 7 ( measured semi-quantitative by Merck pH- indi-cator strips). Finally, the radiopharmaceutical was SepPak
puriied and sterile iltered.
Kit formulation was prepared for a mini batch of 12
vials. We modiied the protocol published for freeze-dried
kit formulation by von Guggenberg (von Guggenberg et al.,2004). Initially, 150 mg of EDDA and 300 mg of tricine were dissolved in 14 mL sterile H2O (in a vial) by heating in water bath. 12 mL of the co-ligand mixture were trans-ferred into 50 ml sterile evacuated closed vial trough 0,20
μ Millipore ilter. 250 μL (1 mg/mL) of HYNIC-TOC so -lution was added with syringe in the vial and mixed.
Final-ly, 150 μL of instant dilution of SnCl2x2H2O in 0,1 M HCl
consisting 240 μg was injected and mixed properly. The
pH of this solution was 4. Finally, the solution was dis-pensed in 12 sterile evacuated vials trough Millipore GV
(low protein binding) ilter in 1 mL volume. The vials were illed with sterile nitrogen and stored frozen at -20 °C.
The labeling procedure of the frozen kit was per-formed after thawing at room temperature, adding 0,5 mL of 0,2N Phosphate buffer and 0,5 mL of 99m
Tc-pertechne-tate. The mixture was incubated for 10 minutes in boiling
water bath. The inal pH was 7. The radiopharmaceutical was SepPak puriied and sterile iltered.
SepPak puriication procedure
A SepPak C-18 mini cartridge was initially activated with 5 mL of 100% ethanol, washed with 5 mL of 0.9% sa-line and dried with 5 mL of air. The radiolabeled solution was passed through the cartridge which was washed after-wards with 5 mL of saline. The labeled peptide was elut-ed from the cartridge with 0.6 mL of 80% ethanol through
Макед. фарм. билт., 57 (1, 2) 65 - 70 (2011)
Quality Control
Radiochemical purity
Instant thin –layer chromatography (ITLC) was per-formed on Silica gel strips with different mobile phases: Methylethylketone (MEK) to determine the percentage of the free 99mTcO
4
- fraction (Rf=1), acetonitryle (ACN) 50%
for the 99mTc-colloid fraction (Rf=0). For the non-peptide
bound 99mTc-co- ligand 0.1M Sodium citrate buffer (pH=5)
(Rf=1) and optionally 0.9% NaCl (Rf=1) was used. Ra-diochemical purity was calculated by supstraction the sum of the impurities from 100%. Radiochromatograms were obtained by Veenstra Radiochromatogram scanner VCS 101, with a software package.
Sterility
The small-batch of frozen kits was tested on sterility, in compliance with the internal GMP Production Protokol.
Biodistribution
For biodistribution studies 7,4 MBq of the
radiophar-maceutical (consisting 1-2 μg of peptide) in a volume of 300 μL was injected into the tail vein of the rats. Animal
scintigrams were obtained after 15, 60 min, 2h and 4h p.i.
in a supine position, with a single-head Simens (e.cam) gamma camera, using a pinhole collimator. Biodistribution within the organs was performed 4h p.i.; blood sample was collected with heparinized syringe by heart puncture, the
animals were sacriiced afterwards and other organs of in -terest (liver, kidneys, spleen, pancreas, adrenals, gut and muscle) extracted. The organs were weighed and radioac-tivity measured respectively in well-type gamma scintilla-tion counter. The results were expressed as percentage of the injected dose per gram of the organ (% ID/g).
Results and Discussion
For the optimization of the labeling protocol, the ra-diochemical impurities obtained by ITLC-SG for the dif-ferent wet labeling protocols were displayed (Table 1) and compared.
The proportion of free pertechnetate was low (0,53% for protocol 1 and 0,36% for protocol 2) as well as the
99mTc-coloid impurities (3.4% and 2.46% respectively).
Al-though the results for the radiochemical impurities were similar in both protocols, the 10 min. boiling water (proto-col 2) was chosen as more convenient, as suggested by von
Table 1. Labeling conditions and radiochemical impurity of the wet labeled 99mTc-EDDA/HYNIC-TOC
Labeling conditions
Radiochemical impurity (mean% ± SD) n=3
99mTcO4 (MEK)
99mTc-RH (ACN 50%)
99mTc-non peptide bound (0.1N Citrate buff.)
70˚C, 30 min. 0.53 ± 0.15 3.4 ± 0.72 5.4 ± 0.82
100˚C, 10 min. 0.36 ± 0.11 2.46 ± 0.76 5.63 ± 1.5
Guggenberg et al (2003). The percentage of the overall ra-diolabeled impurities was less than 10%, accepted as up-per limit and reported by other authors (Plachcinska et al., 2003; von Guggenberg et al., 2004; Melero et al., 2009). For the assessment of non-peptide bound impurities 0.9% NaCl was used elsewhere (Bangard et al. 2000; Plachcins-ka et al.2004). We obtained lower percentage for the above mentioned impurities with 0.9% NaCl (2.8% ±1.06) com-pared to 0.1 M Citrate buffer mobile phase. For more pre-cise detection of peptide related side products, HPLC anal-ysis with radiation detector would be preferable method, but it was not available in our laboratory during this study. Animal biodistribution studies, integrated in our qual-ity control protocol, were carried out with wet labeled kits. The results from the imaging study are presented in Fig. 1.
The radiopharmaceutical was cleared rapidly from the blood, as shown on the 15 min static image, and the most intense radioactivity was observed in the kidneys and
blad-der. The late, 4 h image conirmed the predominant renal
uptake, as reported by Decristoforo et al. (1999). The pro-portion of the injected dose/g in the kidneys was 2, 95% (see Fig. 2), and with lesser extent in the gut (1,16%) and the liver (1,15%). No activity was observed in the thyroid, which was in accordance with the low percentage of free
99mTc-petrechnetate impurity found by ITLC-SG quality
control.
Fig. 2. Biodistribution of 99mTc-EDDA/HYNIC-TOC in
normal Wistar rats 4h p.i (n=2)
Although the wet labeling protocol was conirmed as eficient and reliable procedure for 99mTc labeling of /
HYNIC-TOC, it was more time and labor consuming in comparison with frozen kit labeling. Therefore, prior in-troducing the radiopharmaceutical in the clinical routine, we decided to produce a small kit batch. After the prepa-ration of frozen kits, which were found sterile, their stor-age stability on -20 °C was tested within the period of 6 months. Protocol 2 was used for the radiolabeling, after adding 0,2N phosphate buffer to the mixture. The labeling yield during period observed (expressed as mean of dupli-cate assessment) is displayed on Fig. 3. The range of the
ra-diolabeling yield was between 94,3% and 96,9%. After 6 months, no decrease of radiolabeling yield was observed.
Fig. 3. Storage stability of the frozen kit formulation up to 6 months post production (p.p.= immediately post production)
Fig. 4. Radiochromatograms (ITLC) of 99mTc-EDDA/
HYNIC-TOC kit after 6 months storage at -20 °C: a) MEK (Rf=1) free 99mTc-pertechnetate; b) 50%
ACN (Rf=0) 99mTc-coloid; c) 0.9% NaCl (Rf=1) 99mTc-non peptide bound coligand + free 99m
Макед. фарм. билт., 57 (1, 2) 65 - 70 (2011)
The radiochromatograms obtained from the ITLC strips for the three main radiochemical impurities (Fig. 4.) illustrate the good quality of our radiopharmaceutical and indicate that frozen kit can be a safe alternative to the freeze-dried for small batch in house production.
We could not compare our stability indings with those
published in the literature available, because they are main-ly related to the in house wet labeling procedures (Mel-ero et al., 2009; Pusuwan et al., 2010) or in house freeze dried kits (von Guggenberg et al., 2004; Gandomkar et al., 2006; Tasdelen, 2011). In house production of 99m
Tc-ED-DA/HYNIC-TOC is usually carried out within Coordinat-ed Research Projects or Technical Cooperation Projects of IAEA in labs worldwide due to its restricted availabili-ty. The only commercially released kit so far is Tektrotyd from Radioisotope Centre POLATOM, Poland.
Conclusion
In house kit production, performed in compliance with GMP and GRP regulations within hospital premis-es is sometimpremis-es the only way to introduce novel radiop-harmaceuticas, especially in developing countries. Fol-lowing the proscribed and optimized procedures, we pro-duced 99mTc-EDDA/HYNIC-TOC, a speciic SST recep
-tor targeting agent which fulilled the quality control cri -teria for radiochemical purity, sterility, normal biodistribu-tion and stability. Our future activities will be focused on the clinical application and evaluation on selected group of patients.
Acknowledgements
This work was supported by IAEA Technical Co-op-eration Project MAK3/004 ”Developing of 99mTc labeled peptides and monoclonal antibodies”.
We wish to thank C. Decristophoro and E. von Guggen-berg from the University Clinic for Nuclear Medicine, Innsbruck, Austria for sharing their experience in EDDA/ HYNIC-TOC labeling, and preclinical and clinical evalua-tion of the radiopharmaceutical.
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Резиме
“In house” производство на
99mTc-EDDA/HYNIC-TOC, специфичен насочувачки агенс за соматостатин -
рецепторна сцинтиграфија
Соња Кузмановска, Оливија Васкова, Марина Здравеска Кочовска
Институт за патофизиологија и нуклеарна медицина, Медицински факултет, Универзитет ,,Св. Кирил и Мето
-диј”, Водњанска 17, Скопје, Република Македонија
Клучни зборови: радиофармацевтик, технециум-99m, тир3-октреотид, рецепторна сцинтиграфија, домашно производство, конт
-рола на квалитет.
